A lack of disease-modifying treatment strategies for Alzheimer's disease (AD) results in part from an incomplete understanding of the earliest molecular changes, when there is opportunity for reversal. A body of evidence shows that synaptic dysfunction precedes amyloid-beta (AB) deposition, suggesting that the synaptic compartment is a key site of early pathology. Accurate localization of events to terminals is difficult; therefore, our laboratory has developed methods for flow cytometry analysis of synaptosomes prepared from cryopreserved human and rodent tissue. This technique allows 1) study of populations of individual terminals (5-10,000 particles/sample) and 2) absolute quantification of synaptic AB, phosphorylated tau (p-tau), and lipid and synaptic markers in postmortem human cases and in transgenic disease models. Flow cytometry results will be verified by biochemical assays and confocal and electron microscopy. Our first studies of postmortem AD synaptic terminals showed that ~50% of terminals in AD association cortex are strongly AB-positive. Subsequent experiments have shown that similar AB accumulations occur in the Tg2576 mouse model of AD, and that the AB buildup is accompanied by a significant increase in free cholesterol and GM1 ganglioside. Our general working hypothesis is that synaptic terminal AB accumulation is a key intermediate in synaptic dysfunction prior to loss, and a potential sources or seed for AB deposition in plaques. The specific hypothesis for this project is that cholesterol in lipid raft domains enhances processing of APP to AB; therefore manipulation of terminal cholesterol via ApoE genotype and drug treatment will alter the accumulation of AB and related lipids and peptides, and will affect synaptic function. In the first Specific Aim, our goal is to determine the relationship of synaptic terminal AB to regional AD pathology, disease stage, and apoE isotype in human samples and transgenic mouse models. In the second Specific Aim, we will use dual transgenic mice to determine the effect of apoE isotype on terminals levels of AB, cholesterol and related lipids, and on synaptic function. In addition, we will study the effect of hydrophobic vs. hydrophilic statins on terminal AB accumulation. The third Specific Aim has the goal of identifying the AB peptide species in terminals along with associated changes in signaling pathways, particularly integrin focal adhesion complexes and downstream deregulation of kinase/phosphatase activity. Development of therapeutic strategies for Alzheimer's disease requires a complete understanding of the earliest pathological changes when there is still opportunity for reversal. The present project has the long term goal of identifying potential drug targets and examines pathologic changes in synaptic terminals from Alzheimer's cases and mouse models that are treated with cholesterol lowering drugs. These experiments will help us to understand how cholesterol may contribute to Alzheimer's pathology.